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Alternatives to Laboratory Animals - ATLA

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Toxicity and Cell Density Monitoring in Monolayer and Three-dimensional Cultures with the XTT Assay

Xavier Ponsoda, Maria José Gómez-Lechón and José V. Castell

The application of viability criteria (MTT and XTT tests) to monolayer cultures and immobilised cells in three-dimensional systems was investigated in order to assess cell viability and cell proliferation. The suitability and accuracy of these tests were compared with the conventional criteria (cellular protein and DNA content) used in monolayer cultures for the same purpose. The colorimetric assay based on the metabolic reduction of the tetrazolium salt XTT to a water-soluble formazan proved to be very useful, rapid and sensitive. This automated spectrophotometric enzymatic method, due to its lack of toxicity, also permits repeated nondestructive assays on a single cellular culture for the long-term monitoring of cytotoxicity, cell survival and cell proliferation, and can be performed in 96-well plates with minimal handling. This method could offer a solution for cellular density evaluation in complex cell cultures that do not permit visual examination; it is also the best choice for protein-based, three-dimensional systems such as collagen gels.